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A comparative study of procedures for sheep erythrocyte-human-T-lymphocyte rosette formation
Journal of Immunological Methods, 15 ( 1 9 7 7 ) 2 3 9 - - 2 4 5
239
© E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
A COMPARATIVE S T U D Y OF PROCEDURES FOR SHEEP ERYTHROCYTE-HUMAN-T-LYMPHOCYTE ROSETTE FORMATION
M A R E N L. M A H O W A L D *, B A R R Y S. H A N D W E R G E R , E R S K I N E M. C A P E R T O N E , J R . and S T E V E N D. D O U G L A S
* Department of Medicine, Rheumatology Section, Veterans Adminsitration Hospital, Minneapolis, Minnesota and Department of Medicine, University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota, U.S.A. (Received 30 J u n e 1 9 7 6 , a c c e p t e d 24 N o v e m b e r 1 9 7 6 )
A c o m p a r a t i v e s t u d y o f several p u b l i s h e d m e t h o d s for s h e e p red b l o o d cell-T-lymphoc y t e r o s e t t e f o r m a t i o n was p e r f o r m e d . M a x i m u m S R B C - r o s e t t e f o r m a t i o n o c c u r r e d w i t h A E T t r e a t e d S R B C in m e d i u m s u o o l e m e n t e d w i t h 20% FCS or w i t h u n t r e a t e d SRBC in 100% FCS. P r o l o n g a t i o n of t h e 4°C i n c u b a t i o n p e r i o d f r o m 4 to 18 h e n h a n c e d r o s e t t e f o r m a t i o n . F l u o r e s c e i n d i a c e t a t e staining significantly increased calculated p e r c e n t a g e of r o s e t t e f o r m a t i o n . F l u o r e s c e i n d i a c e t a t e s t a i n i n g significantly increased calculated perc e n t a g e o f r o s e t t e - f o r m i n g l y m p h o c y t e s by alk)wing a c c u r a t e i n d e n t i f i c a t i o n of t h e central l y m p h o c y t e s in morulas.
INTRODUCTION
Spontaneous adherence of sheep erythrocytes (SRBC) to the surface of human peripheral blood T-lymphocytes is a well-documented phenomenon (Wybran and Fudenberg, 1971; Jondal, 1972). Adherence of the sheep red blood cells to the l y m p h o c y t e forms a 'rosette' which can be identified by light microscopy. The precise experimental conditions for optimal or maximal SRBC-rosette formation have not been established. Reported percentages of SRBC-rosette forming lymphocytes in the peripheral blood of normal human subjects varies between 30--90%. These results have involved several variations in procedural technique (Brain, 1970; Coombs, 1970; Lay, 1971; Brain and Gordon, 1971; Winchester, 1974; Hoffman and Kunkel, 1976). Adherence of SRBC to lymphocytes appears to require a two step temperature incubation, with the initial contact at either room temperature or 37°C (Lay, 1971; Mendes, 1973; Hoffman and Kunkel, 1976) and the second stage at 4 °C. Several authors have reported that the age of the SRBC influences adherence (Bentwich, 1973a; Brown and Greaves, 1974) whereas others have found no such effect (Mendes et al., 1973). Centrifugation of the This w o r k was s u p p o r t e d b y NIH USPHS G r a n t s A I - 1 2 4 7 8 - 0 1 and H L - 0 6 3 1 4 - 1 5 , a n d b y a grant from the Minnesota Arthritis Foundation.
240 SRBC and l y m p h o c y t e suspension prior to cell incubation appears to increase SRBC-rosette f or m at i on (Mendes, 1973). Supporting media have included 100% fetal calf serum (Hepburn and Ritts, 1974), plain balanced salt solutions (Mendes, 1973), and synthetic culture media supplemented with different serum types and concentrations (Brain, 1970; Bentwich, 1973a; Mendes, 1973; H o f f m a n and Kunkel, 1976). Several chemical treatments of SRBC have been p e r f o r m e d in an a t t e m p t to increase SRBC-rosette formation. Neuraminidase t r e a t m e n t of SRBC has been r ep o r ted to enhance the SRBC a t t a c h m e n t (Weiner, 1973; Galili and Schlesinger, 1974), however, this remains controversial (Bentwich, 1973b). Trypsinization of SRBC has been r epor t e d to abolish the adherence (Weiner, 1973); in another study, though, it had no effect (Jondal, 1972). Kaplan and Clark (1974) have r e por t ed that t r e a t m e n t of SRBC with 2-amino-ethyisothiouranium bromide (AET), a sulfhydryl reagent results in enhanced binding of SRBC to T-lymphocytes. Surface immunoglobulin positive cells did not rosette with AET-treated sheep er yt hr ocyt es. The a t t a c h m e n t f o r m e d between the SRBC and T cell is easily disrupted and therefore the mixture must be resuspended very gently prior to evaluating rosette f o r m a t i on (Jondal, 1972; Brown and Greaves, 1974). One study, however, has reported t hat slide smears and wet m o u n t preparations of SRBC-rosette suspensions can be made when 100% FCS is used as the supporting medium (Hepburn and Ritts, 1974). The criteria for the det er m i nat i on of the num ber of adherent SRBC necessary for a l y m p h o c y t e to be considered 'rosette-positive' have not been uniform. Some authors have considered only l y m p h o c y t e s with three or more adherent SRBC to be 'positive' for rosette f o r m a t i o n (Bach, 1973) while others have considered any SRBC adherence sufficient (Bentwich, 1973a; H e p b u r n and Ritts, 1974). 'Morulas' or rosettes with so m any adherent SRBC that the central l y m p h o c y t e ( s ) c a n n o t be identified easily occur in most rosette suspensions and make accurate quantitation of rosette-positive l y m p h o c y t e s difficult. Morulas may be either excluded from the counts or included with an estimate of the num ber of rosette-positive and rosette-negative l y m p h o c y t e s which make up an individual conglomerate. Because of the diversity of m e t hods which have been em pl oyed and the variability in the percentage of SRBC-rosetting cells in human peripheral blood that has been reported, we have compared several published SRBCrosette m e t h o d s in a series of parallel experiments. No similar comparison of multiple SRBC-rosette m et hods has been published previously. Published m e t h o d s were modified so t hat incubation periods, cell concentrations and media were comparable. Criteria for determination of rosette positive cells were those r e c o m m e n d e d by the WHO Workshop on Human T and B Cells (Aiuti, 1974). In all m et hods studied, only non-latex containing m o n o n u e l e a r cells with three or more adherent sheep e r y t h r o c y t e s were c o u n ted as 'positive'. L y m p h o c y t e s from normal volunteers were tested for rosette f o r matio n by multiple m et hods on the same day to eliminate the
24 l variation in results that occurs f r om day to day. The methods for SRBC-rosette f o r m a t i o n that were utilized were published by Hepburn and Ritts (1974); Bentwich et al. (1973a); Winchester et al. (1974); Kaplan and Clark (1974); Weiner et al. (1973); Galili and Schlesinger (1974); Bentwich et al. (1973b); and Pang et al. (1974). MATERIALS AND METHODS SRBC were obtained weekly and stored in Alsever's solution. For rosette f o r m a t i o n SRBC were prepared as published in the references cited in table 1 with the following modifications. AET--SRBC were prepared by resuspending 1 volume packed SRBC in 4 volumes of 0.143 M AET (pH 9.0) for 15 min. Trypsin-SRBC were prepared by treating washed SRBC with 0.2% trypsin (Gibco) for 1 h at 37°C. T r e a t m e n t of SRBC with neuraminidase was perf o r m e d as described by Galili and Schlesinger (1974) and by Bentwich et al. (1973b). Aliquots of unt r eat ed ('plain') and chemically modified SRBC were resuspended in 100% FCS and in RPMI 1640 supplemented with 20% FCS (complete medimn). Peripheral blood m o n o n u c l e a r cells were prepared from normal healthy volunteers according to the m e t h o d of B o y u m (1968), resuspended in RPMI 1640 with 50% autologous plasma, incubated with 1--2 × 109 latex particles (Difco, Detroit, Mich., 0.81 pdiameter) for 45 rain at 37°C in a shaking water bath, washed three times, c o u n t e d and resuspended to 2 X 10 ~ non-phagocytosing viable m o n o n u c l e a r cells per cc of 100% FCS and in complete medium. Rosette f o r m a t i o n was p e r f o r m e d according to the previously published m e t h o d s cited above and in Table 1 with the following modifications to give standardized conditions. Sheep e r y t h r o c y t e concent rat i on, m o n o n u c l e a r c e l l c o n cen tr atio n , supporting media, warm incubation time, centrifugation, cold incubation times, m e t h o d of resuspension, staining techniques and counting were identical for each m e t h o d studied. SRBC (50--100 X 106) in 0.5 cc were added to a 12 × 75 mm plastic test tube (Falcon 2054), containing 1 X 106 mo n o n uc l e a r cells in 0.5 cc. The suspension was incubated for 10--15 min at 37°C, centrifuged for 5 rain (50 g) and incubated at 4°C for 4 and 18 h. At the end of the incubation, the suspension was gently rotated by hand to resuspend the cells. E n u m e r a t i o n of rosette forming cells was perf o r med using bot h wet m o u n t preparations and the slide m e t h o d described by Hepburn and Ritts (1974). For the wet m o u n t rosette preparations, one drop o f trypan blue (0.2% in normal saline) or one drop of a 1/1000 dilution of fluorescein diacetate (Sigma, St. Louis, Mo.) was added to the cell suspension (Ramasamy, 1974), the cells placed under a disposable coverslip, and c o u n ted in a h e m o c y t o m e t e r , using a Zeiss P h o t o m i c r o s c o p e II with an Ozam HBO 200 W m e r cur y arc light source equipped with an epi-illuminator for fluorescence and a halogen light source for transmitted phase microscopy. In some experiments rosettes were stabilized by glutaraldehyde treat-
242 TABLE 1 Percentage o f S R B C - l y m p h o c y t e r o s e t t e f o r m a t i o n in n o r m a l h u m a n s . Slide m e t h o d
Ref.
1. Plain SRBC b
H e p b u r n and Ritts, 1974
2. A E T - S R B C c 3. T r y p s i n - S R B C d Wet m o u n t preparation
3. FCS-SRBC e
18 h 4~'C 4. A E T - S R B C
13 10 7
Percent rosette positive cells a Ave
Range
60 62 46
33--78 20--93 19--79
T r y p a n blue stain
Ref. 1. T r y p s i n - S R B C 18 h 4°C 2. PIain-SRBC 4 h 4°C 18 h 4°C
No
Weiner, 1973 Bentwich, 1973a Winchester, 1974 H e p b u r n and Ritts, 1974 Kaplan and Clark, 1974
4 h 4°C 18 h 4°C
F l u o r e s c e i n diacetate stain
Percent rosette positive cells
Percent rosette positive cells
Ave
Range
No
6
41
22--51
9
46
10
No
Ave
Range
6
66
46--69
34--51
8
55
43--66
41
20--58
9
66
44--75
8
53
45--62
8
74
65--85
3 10
38 40
33--46 19--67
3 10
72 79
68--76 63--88
P e r c e n t positive cells
Surface i m m u n o g l o b u l i n Aggregate b i n d i n g L a t e x ingestion
No 19 7 16
Ave 18 13 9
Range 6--24 3--23 1--20
a In all m e t h o d s s t u d i e d , r o s e t t e positive refers to the p e r c e n t a g e of n o n - l a t e x c o n t a i n i n g m o n o n u c l e a r cells to w h i c h 3 or m o r e s h e e p e r y t h r o c y t e s were a d h e r e n t (Aiuti et al., 1974). A m i n i m u m o f 200 cells were c o u n t e d per assay. b Plain-SRBC are w a s h e d SRBC. c A E T - S R B C are A E T - t r e a t e d SRBC. d T r y p s i n - S R B C are t r y p s i n i z e d SRBC. e FCS-SRBC are SRBC washed and r e s u s p e n d e d in 100% fetal calf serum. ment according
to the method
of Pang (1974).
R o s e t t e s w e r e c o u n t e d as p o s i t i v e o n l y if t h r e e o r m o r e S R B C w e r e a d h e rent to a non-latex-containing mononuclear cell, according to the recommen-
243 dations o f the WHO Workshop (Aiuti, 1974). A minimum of 200 cells per assay were counted. Clumps of SRBC (morulas) were not c o u n t e d as rosettes unless the central l y m p h o c y t e ( s ) could be clearly identified. Clumps for four or more m o n o n u c l e a r cells were excluded from counting. Staining of l y m p h o c y t e surface immunoglobulin and binding of aggregated human immunoglobulin was p e r f o r m e d as described by Dickler and Kunkel (1972). In several experiments, triple labeling procedures were carried out to d e m o n s t r a t e that rosette f o r m a t i o n was not the result of adherence of sheep e r y t h r o c y t e s to latex ingesting cells or cells bearing surface immunoglobulin in the various test conditions employed. After the incubation with latex particles as described above, m o n o n u c l e a r cells were washed three times with cold phoshate buffered saline with 2% bovine serum albumin, pH 7.3 (PBSBSA). One million cells were resuspended in 50 ~ PBS-BSA, 50 ~ of a 1 : 1 dilution of R h o dam i ne conjugated F(ab')2 fragment of rabbit anti F(ab')2 of human IgG (Cappel) was added and the mixture incubated for 30 min at 4°C. After two washes with cold PBS-BSA the cells were resuspended in complete m e d iu m or 100% fetal calf serum for use in rosette formation. Results are expressed as the arithmetic mean and range for each method. Statistical comparisons were made using the paired observation t test. Where values were significantly different a p value is indicated. RESULTS AND DISCUSSION The design of this study has allowed direct comparison of several m et hods for rosette f o r m a t i o n with standardized l y m p h o c y t e , SRBC, and serum concentrations, l y m p h o c y t e to SRBC ratio, incubation periods and criteria for evaluating rosette positive l ym phoc yt e s . As illustrated in table 1, the slide m e t h o d showed an average of 60% of l y m p h o c y t e s forming rosettes with plain SRBC, 62% with AET treated SRBC and 46% with trypsinized SRBC. These values are lower than those reported b y He pbur n and Ritts (1974) who included as 'rosette-positive' l y m p h o c y t e s with one or two adherent SRBC. In our hands, the slide m e t h o d results in the non-uniform distribution of cells and rosettes. A significant variability occurred in the qua nt i t a t i on of SRBC-rosette f o r m a t i o n depending on which area of the slide was read. Variability was present even when an area of the slide containing evenly dispersed RBC was selected. The percentages of SRBC-rosette forming l y m p h o c y t e s obtained using the various wet m o u n t preparations are recorded in table 1. T r e a t m e n t of SRBC with trypsin did not increase rosette f o r m a t i o n above that observed with plain SRBC {18 h 4°C incubation of rosettes). Glutaraldehyde fixation of rosette suspensions pr oduc e d an increase in the calculated percentage of rosette f o r m a t i o n (glutaraldehyde treated = 80% rosette positive), but this increase was specious and largely due to the form at i on of large clumps of non-rosetted l y m p h o c y t e s which were excluded from the count (Vida supra). Fluorescein diacetate staining made possible the accurate identification
244 and q u a n t i t a t i o n o f t h e c e n t r a l l y m p h o c y t e ( s ) in m o r u l a s and t h e r e b y r e s u l t e d in a significant increase (P < 0 . 0 0 5 ) in t h e p e r c e n t a g e of r o s e t t e form a t i o n f o r all t h e w e t m o u n t p r e p a r a t i o n s studied. In a d d i t i o n , t h e use of f l u o r e s c e i n d i a c e t a t e resulted in i m p r o v e d r e p r o d u c i b i l i t y and d e c r e a s e d t h e t i m e r e q u i r e d f o r c o u n t i n g each sample. Increasing t h e 4°C i n c u b a t i o n f r o m 4 h t o 18 h e n h a n c e d r o s e t t e f o r m a t i o n f o r plain (55 t o 66%, P < 0 . 0 0 5 ) or A E T - t r e a t e d S R B C (72 to 77%, P < 0.05). P r e t r e a t m e n t of s h e e p e r y t h r o c y t e s w i t h n e u r a m i n i d a s e for 1 h at 37°C r e s u l t e d in t h e f o r m a t i o n of stable r o s e t t e s a f t e r i n c u b a t i o n w i t h l y m p h o c y t e s f o r f o u r h o u r s at 4 °C. T h e s e r o s e t t e s resisted v o r t e x i n g b u t t h e perc e n t a g e r o s e t t e f o r m a t i o n was n o t increased w h e n c o m p a r e d t o u n t r e a t e d S R B C (46% f o r plain SRBC and 40% f o r n e u r a m i n i d a s e t r e a t e d SRBC). T r e a t m e n t of S R B C w i t h A E T increased r o s e t t e f o r m a t i o n f r o m 55 to 72% a f t e r 4 h at 4°C and f r o m 64 to 79% ( P < 0 . 0 0 5 ) a f t e r 18 h at 4°C, and d e c r e a s e d the range o f values o b t a i n e d . By c o m p a r i n g s o m e individual values t h e m e r i t s o f these o b s e r v a t i o n s are f u r t h e r d e m o n s t r a t e d . F o r e x a m p l e , in t w o s a m p l e s w h i c h gave low values w i t h plain SRBC a f t e r a 4 h 4°C i n c u b a t i o n (43 and 50%), t h e p e r c e n t rosette f o r m a t i o n increased to 58% a n d 75% w i t h an 18 h 4°C i n c u b a t i o n . T h e s e values w e r e f u r t h e r increased to 80% and 84% w h e n A E T t r e a t e d SRBC w e r e used (18 h, 4°C i n c u b a t i o n ) . This suggests t h a t s o m e n o r m a l individuals m a y have a subset of T - l y m p h o c y t e s t h a t requires m o r e t i m e f o r SRBC a d h e r e n c e or t r e a t m e n t of t h e SRBC w i t h A E T to a u g m e n t binding. TABLE 2 Triple markers. Surface Ig only
No marker
Rosette with 3 or more RBC
Rosette with less than 3 RBC
Latex positive cells
11%
10%
65.5% 0.5% SIg and latex positive
11.5% 1% also SIg%
2%
r
Sample A Plain-SRBC 18h4~C
FCS-SRBC 18 h 4°C Sample B Plain SRBC 18h4°C FCS-SRBC 18h4°C
8.5%
7%
73.5%
7.5%
]7%
63%
7.5%
8.5%
77%
8.5%
7.5% 0.5% also SIg positive 4% 1% also SIg positive
2.5%
2.5%
3%
245 T h e use o f 100% f e t a l calf s e r u m as t h e s u p p o r t i n g m e d i u m f o r 18 h w e t p r e p a r a t i o n s of plain S R B C - r o s e t t e s , gave p e r c e n t a g e s o f r o s e t t e f o r m a t i o n t h a t are c o m p a r a b l e to t h o s e o b t a i n e d using A E T t r e a t e d SRBC, and d o e s n o t require t h e e x t r a t i m e f o r A E T t r e a t m e n t f o r SRBC. By the use of triple labeling e x p e r i m e n t s , we d e m o n s t r a t e d t h a t increased r o s e t t e f o r m a t i o n w i t h c o m p l e t e m e d i a and A E T - t r e a t e d SRBC or 100% FCS and plain S R B C was n o t d u e to the a d h e r e n c e o f s h e e p e r y t h r o c y t e s to latex ingesting or surface i m m u n o g l o b u l i n bearing cells. R a r e l y ( 1 / 8 0 0 ) a large m o n o n u c l e a r cell was seen which c o n t a i n s latex particles, bears surface i m m u n o g l o b u l i n and was s u r r o u n d e d b y d e n s e l y a d h e r e n t sheep e r y t h r o c y t e s . This has been p r e v i o u s l y n o t e d b y (',lark and K a p l a n w i t h t h e use o f A E T t r e a t e d s h e e p e r y t h r o c y t e s . An e x a m p l e o f a triple m a r k e r e x p e r i m e n t f o r t w o n o r m a l c o n t r o l s using plain sheep e r y t h r o c y t e s in c o m p l e t e m e d i a a n d 100% fetal calf s e r u m is illustrated w i t h individual results in t a b l e 2. In c o n c l u s i o n we have d e m o n s t r a t e d t h a t o f the m e t h o d s t e s t e d , w e t m o u n t p r e p a r a t i o n s o f S R B C - r o s e t t e s using 100% fetal calf s e r u m and plain S R B C or c o m p l e t e m e d i u m and A E T t r e a t e d S R B C p r o d u c e d the m a x i m u m p e r c e n t a g e of S R B C - r o s e t t e f o r m a t i o n . P r o l o n g i n g the 4°C i n c u b a t i o n f r o m 4 to 18 h significantly increased S R B C - r o s e t t e f o r m a t i o n . F l u o r e s c e i n diacet a t e staining, b y allowing a c c u r a t e i d e n t i f i c a t i o n and q u a n t i t a t i o n o f central l y m p h o c y t e ( s ) significantly increased the calculated p e r c e n t a g e o f SRBCrosette formation. REFERENCES Aiuti, E et al., 1974, Scand. J. Immunol. 3. 521. Bach, J.F., 1973, Trans. Rev. 16,196. Bentwich, Z. et al., 1973a, Clinl Immunol. Immunopathol. 1, 511. Bentwich, Z. et al., 1973b, J. Exp. Med. 137, 1532. Boyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl), 97. Brain, P. et al., 1970, Clin. Exp. Immunol. 6, 681. Brain, P. and J. Gorden, 1971, J. Clin. Exp. Immunol. 8,441. Brown, G. and M.F. Greaves, 1974, Eur. J. Immunol. 4,302. Coombs, R.R.A. et al., 1970, Int. Arch. Allergy 39,658. Dick]er, H.B. and H.G. Kunkel, 1972, J. Exp. Med. 136, 191. Galili, U. and M. Schlesinger, 1974, J. Immunol. 112, 1628. Hepburn, B. and R.E. Ritts, 1974, Mayo Clin. Proc. 49, 866. Hoffman, T. and H.G. Kunkel, 1976, submitted for publication. Jondal, M. et al., 1972, J. Exp. Med. 136,207. Kaplan, M.E. and C. Clark, 1974, J. Immunol. Methods 5, 131. Lay, W.H. et al., 1970, Int. Arch. Allergy 39,658. Mendes, N.F. et al., 1973, J. Immunol. 111,860. Pang, G.T.M. et al., 1974, J. Immunol. Methods, 4, 41. Ramasamy, R., 1974, J. Immunol. Methods 5, 305. Weiner, M.S. et al., 1973, Blood 42,939. Winchester, R.J. et al., 1974, J. Clin. Invest. 54, 1082. Winchester, R.J. et al., 1975, J. Immunol. 11,1, 1210. Wybran, J.H. and H.H. Fudenberg, 1971, Trans. Ass. Am. Phys. 84, 239.
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A COMPARATIVE S T U D Y OF PROCEDURES FOR SHEEP ERYTHROCYTE-HUMAN-T-LYMPHOCYTE ROSETTE FORMATION
M A R E N L. M A H O W A L D *, B A R R Y S. H A N D W E R G E R , E R S K I N E M. C A P E R T O N E , J R . and S T E V E N D. D O U G L A S
* Department of Medicine, Rheumatology Section, Veterans Adminsitration Hospital, Minneapolis, Minnesota and Department of Medicine, University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota, U.S.A. (Received 30 J u n e 1 9 7 6 , a c c e p t e d 24 N o v e m b e r 1 9 7 6 )
A c o m p a r a t i v e s t u d y o f several p u b l i s h e d m e t h o d s for s h e e p red b l o o d cell-T-lymphoc y t e r o s e t t e f o r m a t i o n was p e r f o r m e d . M a x i m u m S R B C - r o s e t t e f o r m a t i o n o c c u r r e d w i t h A E T t r e a t e d S R B C in m e d i u m s u o o l e m e n t e d w i t h 20% FCS or w i t h u n t r e a t e d SRBC in 100% FCS. P r o l o n g a t i o n of t h e 4°C i n c u b a t i o n p e r i o d f r o m 4 to 18 h e n h a n c e d r o s e t t e f o r m a t i o n . F l u o r e s c e i n d i a c e t a t e staining significantly increased calculated p e r c e n t a g e of r o s e t t e f o r m a t i o n . F l u o r e s c e i n d i a c e t a t e s t a i n i n g significantly increased calculated perc e n t a g e o f r o s e t t e - f o r m i n g l y m p h o c y t e s by alk)wing a c c u r a t e i n d e n t i f i c a t i o n of t h e central l y m p h o c y t e s in morulas.
INTRODUCTION
Spontaneous adherence of sheep erythrocytes (SRBC) to the surface of human peripheral blood T-lymphocytes is a well-documented phenomenon (Wybran and Fudenberg, 1971; Jondal, 1972). Adherence of the sheep red blood cells to the l y m p h o c y t e forms a 'rosette' which can be identified by light microscopy. The precise experimental conditions for optimal or maximal SRBC-rosette formation have not been established. Reported percentages of SRBC-rosette forming lymphocytes in the peripheral blood of normal human subjects varies between 30--90%. These results have involved several variations in procedural technique (Brain, 1970; Coombs, 1970; Lay, 1971; Brain and Gordon, 1971; Winchester, 1974; Hoffman and Kunkel, 1976). Adherence of SRBC to lymphocytes appears to require a two step temperature incubation, with the initial contact at either room temperature or 37°C (Lay, 1971; Mendes, 1973; Hoffman and Kunkel, 1976) and the second stage at 4 °C. Several authors have reported that the age of the SRBC influences adherence (Bentwich, 1973a; Brown and Greaves, 1974) whereas others have found no such effect (Mendes et al., 1973). Centrifugation of the This w o r k was s u p p o r t e d b y NIH USPHS G r a n t s A I - 1 2 4 7 8 - 0 1 and H L - 0 6 3 1 4 - 1 5 , a n d b y a grant from the Minnesota Arthritis Foundation.
240 SRBC and l y m p h o c y t e suspension prior to cell incubation appears to increase SRBC-rosette f or m at i on (Mendes, 1973). Supporting media have included 100% fetal calf serum (Hepburn and Ritts, 1974), plain balanced salt solutions (Mendes, 1973), and synthetic culture media supplemented with different serum types and concentrations (Brain, 1970; Bentwich, 1973a; Mendes, 1973; H o f f m a n and Kunkel, 1976). Several chemical treatments of SRBC have been p e r f o r m e d in an a t t e m p t to increase SRBC-rosette formation. Neuraminidase t r e a t m e n t of SRBC has been r ep o r ted to enhance the SRBC a t t a c h m e n t (Weiner, 1973; Galili and Schlesinger, 1974), however, this remains controversial (Bentwich, 1973b). Trypsinization of SRBC has been r epor t e d to abolish the adherence (Weiner, 1973); in another study, though, it had no effect (Jondal, 1972). Kaplan and Clark (1974) have r e por t ed that t r e a t m e n t of SRBC with 2-amino-ethyisothiouranium bromide (AET), a sulfhydryl reagent results in enhanced binding of SRBC to T-lymphocytes. Surface immunoglobulin positive cells did not rosette with AET-treated sheep er yt hr ocyt es. The a t t a c h m e n t f o r m e d between the SRBC and T cell is easily disrupted and therefore the mixture must be resuspended very gently prior to evaluating rosette f o r m a t i on (Jondal, 1972; Brown and Greaves, 1974). One study, however, has reported t hat slide smears and wet m o u n t preparations of SRBC-rosette suspensions can be made when 100% FCS is used as the supporting medium (Hepburn and Ritts, 1974). The criteria for the det er m i nat i on of the num ber of adherent SRBC necessary for a l y m p h o c y t e to be considered 'rosette-positive' have not been uniform. Some authors have considered only l y m p h o c y t e s with three or more adherent SRBC to be 'positive' for rosette f o r m a t i o n (Bach, 1973) while others have considered any SRBC adherence sufficient (Bentwich, 1973a; H e p b u r n and Ritts, 1974). 'Morulas' or rosettes with so m any adherent SRBC that the central l y m p h o c y t e ( s ) c a n n o t be identified easily occur in most rosette suspensions and make accurate quantitation of rosette-positive l y m p h o c y t e s difficult. Morulas may be either excluded from the counts or included with an estimate of the num ber of rosette-positive and rosette-negative l y m p h o c y t e s which make up an individual conglomerate. Because of the diversity of m e t hods which have been em pl oyed and the variability in the percentage of SRBC-rosetting cells in human peripheral blood that has been reported, we have compared several published SRBCrosette m e t h o d s in a series of parallel experiments. No similar comparison of multiple SRBC-rosette m et hods has been published previously. Published m e t h o d s were modified so t hat incubation periods, cell concentrations and media were comparable. Criteria for determination of rosette positive cells were those r e c o m m e n d e d by the WHO Workshop on Human T and B Cells (Aiuti, 1974). In all m et hods studied, only non-latex containing m o n o n u e l e a r cells with three or more adherent sheep e r y t h r o c y t e s were c o u n ted as 'positive'. L y m p h o c y t e s from normal volunteers were tested for rosette f o r matio n by multiple m et hods on the same day to eliminate the
24 l variation in results that occurs f r om day to day. The methods for SRBC-rosette f o r m a t i o n that were utilized were published by Hepburn and Ritts (1974); Bentwich et al. (1973a); Winchester et al. (1974); Kaplan and Clark (1974); Weiner et al. (1973); Galili and Schlesinger (1974); Bentwich et al. (1973b); and Pang et al. (1974). MATERIALS AND METHODS SRBC were obtained weekly and stored in Alsever's solution. For rosette f o r m a t i o n SRBC were prepared as published in the references cited in table 1 with the following modifications. AET--SRBC were prepared by resuspending 1 volume packed SRBC in 4 volumes of 0.143 M AET (pH 9.0) for 15 min. Trypsin-SRBC were prepared by treating washed SRBC with 0.2% trypsin (Gibco) for 1 h at 37°C. T r e a t m e n t of SRBC with neuraminidase was perf o r m e d as described by Galili and Schlesinger (1974) and by Bentwich et al. (1973b). Aliquots of unt r eat ed ('plain') and chemically modified SRBC were resuspended in 100% FCS and in RPMI 1640 supplemented with 20% FCS (complete medimn). Peripheral blood m o n o n u c l e a r cells were prepared from normal healthy volunteers according to the m e t h o d of B o y u m (1968), resuspended in RPMI 1640 with 50% autologous plasma, incubated with 1--2 × 109 latex particles (Difco, Detroit, Mich., 0.81 pdiameter) for 45 rain at 37°C in a shaking water bath, washed three times, c o u n t e d and resuspended to 2 X 10 ~ non-phagocytosing viable m o n o n u c l e a r cells per cc of 100% FCS and in complete medium. Rosette f o r m a t i o n was p e r f o r m e d according to the previously published m e t h o d s cited above and in Table 1 with the following modifications to give standardized conditions. Sheep e r y t h r o c y t e concent rat i on, m o n o n u c l e a r c e l l c o n cen tr atio n , supporting media, warm incubation time, centrifugation, cold incubation times, m e t h o d of resuspension, staining techniques and counting were identical for each m e t h o d studied. SRBC (50--100 X 106) in 0.5 cc were added to a 12 × 75 mm plastic test tube (Falcon 2054), containing 1 X 106 mo n o n uc l e a r cells in 0.5 cc. The suspension was incubated for 10--15 min at 37°C, centrifuged for 5 rain (50 g) and incubated at 4°C for 4 and 18 h. At the end of the incubation, the suspension was gently rotated by hand to resuspend the cells. E n u m e r a t i o n of rosette forming cells was perf o r med using bot h wet m o u n t preparations and the slide m e t h o d described by Hepburn and Ritts (1974). For the wet m o u n t rosette preparations, one drop o f trypan blue (0.2% in normal saline) or one drop of a 1/1000 dilution of fluorescein diacetate (Sigma, St. Louis, Mo.) was added to the cell suspension (Ramasamy, 1974), the cells placed under a disposable coverslip, and c o u n ted in a h e m o c y t o m e t e r , using a Zeiss P h o t o m i c r o s c o p e II with an Ozam HBO 200 W m e r cur y arc light source equipped with an epi-illuminator for fluorescence and a halogen light source for transmitted phase microscopy. In some experiments rosettes were stabilized by glutaraldehyde treat-
242 TABLE 1 Percentage o f S R B C - l y m p h o c y t e r o s e t t e f o r m a t i o n in n o r m a l h u m a n s . Slide m e t h o d
Ref.
1. Plain SRBC b
H e p b u r n and Ritts, 1974
2. A E T - S R B C c 3. T r y p s i n - S R B C d Wet m o u n t preparation
3. FCS-SRBC e
18 h 4~'C 4. A E T - S R B C
13 10 7
Percent rosette positive cells a Ave
Range
60 62 46
33--78 20--93 19--79
T r y p a n blue stain
Ref. 1. T r y p s i n - S R B C 18 h 4°C 2. PIain-SRBC 4 h 4°C 18 h 4°C
No
Weiner, 1973 Bentwich, 1973a Winchester, 1974 H e p b u r n and Ritts, 1974 Kaplan and Clark, 1974
4 h 4°C 18 h 4°C
F l u o r e s c e i n diacetate stain
Percent rosette positive cells
Percent rosette positive cells
Ave
Range
No
6
41
22--51
9
46
10
No
Ave
Range
6
66
46--69
34--51
8
55
43--66
41
20--58
9
66
44--75
8
53
45--62
8
74
65--85
3 10
38 40
33--46 19--67
3 10
72 79
68--76 63--88
P e r c e n t positive cells
Surface i m m u n o g l o b u l i n Aggregate b i n d i n g L a t e x ingestion
No 19 7 16
Ave 18 13 9
Range 6--24 3--23 1--20
a In all m e t h o d s s t u d i e d , r o s e t t e positive refers to the p e r c e n t a g e of n o n - l a t e x c o n t a i n i n g m o n o n u c l e a r cells to w h i c h 3 or m o r e s h e e p e r y t h r o c y t e s were a d h e r e n t (Aiuti et al., 1974). A m i n i m u m o f 200 cells were c o u n t e d per assay. b Plain-SRBC are w a s h e d SRBC. c A E T - S R B C are A E T - t r e a t e d SRBC. d T r y p s i n - S R B C are t r y p s i n i z e d SRBC. e FCS-SRBC are SRBC washed and r e s u s p e n d e d in 100% fetal calf serum. ment according
to the method
of Pang (1974).
R o s e t t e s w e r e c o u n t e d as p o s i t i v e o n l y if t h r e e o r m o r e S R B C w e r e a d h e rent to a non-latex-containing mononuclear cell, according to the recommen-
243 dations o f the WHO Workshop (Aiuti, 1974). A minimum of 200 cells per assay were counted. Clumps of SRBC (morulas) were not c o u n t e d as rosettes unless the central l y m p h o c y t e ( s ) could be clearly identified. Clumps for four or more m o n o n u c l e a r cells were excluded from counting. Staining of l y m p h o c y t e surface immunoglobulin and binding of aggregated human immunoglobulin was p e r f o r m e d as described by Dickler and Kunkel (1972). In several experiments, triple labeling procedures were carried out to d e m o n s t r a t e that rosette f o r m a t i o n was not the result of adherence of sheep e r y t h r o c y t e s to latex ingesting cells or cells bearing surface immunoglobulin in the various test conditions employed. After the incubation with latex particles as described above, m o n o n u c l e a r cells were washed three times with cold phoshate buffered saline with 2% bovine serum albumin, pH 7.3 (PBSBSA). One million cells were resuspended in 50 ~ PBS-BSA, 50 ~ of a 1 : 1 dilution of R h o dam i ne conjugated F(ab')2 fragment of rabbit anti F(ab')2 of human IgG (Cappel) was added and the mixture incubated for 30 min at 4°C. After two washes with cold PBS-BSA the cells were resuspended in complete m e d iu m or 100% fetal calf serum for use in rosette formation. Results are expressed as the arithmetic mean and range for each method. Statistical comparisons were made using the paired observation t test. Where values were significantly different a p value is indicated. RESULTS AND DISCUSSION The design of this study has allowed direct comparison of several m et hods for rosette f o r m a t i o n with standardized l y m p h o c y t e , SRBC, and serum concentrations, l y m p h o c y t e to SRBC ratio, incubation periods and criteria for evaluating rosette positive l ym phoc yt e s . As illustrated in table 1, the slide m e t h o d showed an average of 60% of l y m p h o c y t e s forming rosettes with plain SRBC, 62% with AET treated SRBC and 46% with trypsinized SRBC. These values are lower than those reported b y He pbur n and Ritts (1974) who included as 'rosette-positive' l y m p h o c y t e s with one or two adherent SRBC. In our hands, the slide m e t h o d results in the non-uniform distribution of cells and rosettes. A significant variability occurred in the qua nt i t a t i on of SRBC-rosette f o r m a t i o n depending on which area of the slide was read. Variability was present even when an area of the slide containing evenly dispersed RBC was selected. The percentages of SRBC-rosette forming l y m p h o c y t e s obtained using the various wet m o u n t preparations are recorded in table 1. T r e a t m e n t of SRBC with trypsin did not increase rosette f o r m a t i o n above that observed with plain SRBC {18 h 4°C incubation of rosettes). Glutaraldehyde fixation of rosette suspensions pr oduc e d an increase in the calculated percentage of rosette f o r m a t i o n (glutaraldehyde treated = 80% rosette positive), but this increase was specious and largely due to the form at i on of large clumps of non-rosetted l y m p h o c y t e s which were excluded from the count (Vida supra). Fluorescein diacetate staining made possible the accurate identification
244 and q u a n t i t a t i o n o f t h e c e n t r a l l y m p h o c y t e ( s ) in m o r u l a s and t h e r e b y r e s u l t e d in a significant increase (P < 0 . 0 0 5 ) in t h e p e r c e n t a g e of r o s e t t e form a t i o n f o r all t h e w e t m o u n t p r e p a r a t i o n s studied. In a d d i t i o n , t h e use of f l u o r e s c e i n d i a c e t a t e resulted in i m p r o v e d r e p r o d u c i b i l i t y and d e c r e a s e d t h e t i m e r e q u i r e d f o r c o u n t i n g each sample. Increasing t h e 4°C i n c u b a t i o n f r o m 4 h t o 18 h e n h a n c e d r o s e t t e f o r m a t i o n f o r plain (55 t o 66%, P < 0 . 0 0 5 ) or A E T - t r e a t e d S R B C (72 to 77%, P < 0.05). P r e t r e a t m e n t of s h e e p e r y t h r o c y t e s w i t h n e u r a m i n i d a s e for 1 h at 37°C r e s u l t e d in t h e f o r m a t i o n of stable r o s e t t e s a f t e r i n c u b a t i o n w i t h l y m p h o c y t e s f o r f o u r h o u r s at 4 °C. T h e s e r o s e t t e s resisted v o r t e x i n g b u t t h e perc e n t a g e r o s e t t e f o r m a t i o n was n o t increased w h e n c o m p a r e d t o u n t r e a t e d S R B C (46% f o r plain SRBC and 40% f o r n e u r a m i n i d a s e t r e a t e d SRBC). T r e a t m e n t of S R B C w i t h A E T increased r o s e t t e f o r m a t i o n f r o m 55 to 72% a f t e r 4 h at 4°C and f r o m 64 to 79% ( P < 0 . 0 0 5 ) a f t e r 18 h at 4°C, and d e c r e a s e d the range o f values o b t a i n e d . By c o m p a r i n g s o m e individual values t h e m e r i t s o f these o b s e r v a t i o n s are f u r t h e r d e m o n s t r a t e d . F o r e x a m p l e , in t w o s a m p l e s w h i c h gave low values w i t h plain SRBC a f t e r a 4 h 4°C i n c u b a t i o n (43 and 50%), t h e p e r c e n t rosette f o r m a t i o n increased to 58% a n d 75% w i t h an 18 h 4°C i n c u b a t i o n . T h e s e values w e r e f u r t h e r increased to 80% and 84% w h e n A E T t r e a t e d SRBC w e r e used (18 h, 4°C i n c u b a t i o n ) . This suggests t h a t s o m e n o r m a l individuals m a y have a subset of T - l y m p h o c y t e s t h a t requires m o r e t i m e f o r SRBC a d h e r e n c e or t r e a t m e n t of t h e SRBC w i t h A E T to a u g m e n t binding. TABLE 2 Triple markers. Surface Ig only
No marker
Rosette with 3 or more RBC
Rosette with less than 3 RBC
Latex positive cells
11%
10%
65.5% 0.5% SIg and latex positive
11.5% 1% also SIg%
2%
r
Sample A Plain-SRBC 18h4~C
FCS-SRBC 18 h 4°C Sample B Plain SRBC 18h4°C FCS-SRBC 18h4°C
8.5%
7%
73.5%
7.5%
]7%
63%
7.5%
8.5%
77%
8.5%
7.5% 0.5% also SIg positive 4% 1% also SIg positive
2.5%
2.5%
3%
245 T h e use o f 100% f e t a l calf s e r u m as t h e s u p p o r t i n g m e d i u m f o r 18 h w e t p r e p a r a t i o n s of plain S R B C - r o s e t t e s , gave p e r c e n t a g e s o f r o s e t t e f o r m a t i o n t h a t are c o m p a r a b l e to t h o s e o b t a i n e d using A E T t r e a t e d SRBC, and d o e s n o t require t h e e x t r a t i m e f o r A E T t r e a t m e n t f o r SRBC. By the use of triple labeling e x p e r i m e n t s , we d e m o n s t r a t e d t h a t increased r o s e t t e f o r m a t i o n w i t h c o m p l e t e m e d i a and A E T - t r e a t e d SRBC or 100% FCS and plain S R B C was n o t d u e to the a d h e r e n c e o f s h e e p e r y t h r o c y t e s to latex ingesting or surface i m m u n o g l o b u l i n bearing cells. R a r e l y ( 1 / 8 0 0 ) a large m o n o n u c l e a r cell was seen which c o n t a i n s latex particles, bears surface i m m u n o g l o b u l i n and was s u r r o u n d e d b y d e n s e l y a d h e r e n t sheep e r y t h r o c y t e s . This has been p r e v i o u s l y n o t e d b y (',lark and K a p l a n w i t h t h e use o f A E T t r e a t e d s h e e p e r y t h r o c y t e s . An e x a m p l e o f a triple m a r k e r e x p e r i m e n t f o r t w o n o r m a l c o n t r o l s using plain sheep e r y t h r o c y t e s in c o m p l e t e m e d i a a n d 100% fetal calf s e r u m is illustrated w i t h individual results in t a b l e 2. In c o n c l u s i o n we have d e m o n s t r a t e d t h a t o f the m e t h o d s t e s t e d , w e t m o u n t p r e p a r a t i o n s o f S R B C - r o s e t t e s using 100% fetal calf s e r u m and plain S R B C or c o m p l e t e m e d i u m and A E T t r e a t e d S R B C p r o d u c e d the m a x i m u m p e r c e n t a g e of S R B C - r o s e t t e f o r m a t i o n . P r o l o n g i n g the 4°C i n c u b a t i o n f r o m 4 to 18 h significantly increased S R B C - r o s e t t e f o r m a t i o n . F l u o r e s c e i n diacet a t e staining, b y allowing a c c u r a t e i d e n t i f i c a t i o n and q u a n t i t a t i o n o f central l y m p h o c y t e ( s ) significantly increased the calculated p e r c e n t a g e o f SRBCrosette formation. REFERENCES Aiuti, E et al., 1974, Scand. J. Immunol. 3. 521. Bach, J.F., 1973, Trans. Rev. 16,196. Bentwich, Z. et al., 1973a, Clinl Immunol. Immunopathol. 1, 511. Bentwich, Z. et al., 1973b, J. Exp. Med. 137, 1532. Boyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl), 97. Brain, P. et al., 1970, Clin. Exp. Immunol. 6, 681. Brain, P. and J. Gorden, 1971, J. Clin. Exp. Immunol. 8,441. Brown, G. and M.F. Greaves, 1974, Eur. J. Immunol. 4,302. Coombs, R.R.A. et al., 1970, Int. Arch. Allergy 39,658. Dick]er, H.B. and H.G. Kunkel, 1972, J. Exp. Med. 136, 191. Galili, U. and M. Schlesinger, 1974, J. Immunol. 112, 1628. Hepburn, B. and R.E. Ritts, 1974, Mayo Clin. Proc. 49, 866. Hoffman, T. and H.G. Kunkel, 1976, submitted for publication. Jondal, M. et al., 1972, J. Exp. Med. 136,207. Kaplan, M.E. and C. Clark, 1974, J. Immunol. Methods 5, 131. Lay, W.H. et al., 1970, Int. Arch. Allergy 39,658. Mendes, N.F. et al., 1973, J. Immunol. 111,860. Pang, G.T.M. et al., 1974, J. Immunol. Methods, 4, 41. Ramasamy, R., 1974, J. Immunol. Methods 5, 305. Weiner, M.S. et al., 1973, Blood 42,939. Winchester, R.J. et al., 1974, J. Clin. Invest. 54, 1082. Winchester, R.J. et al., 1975, J. Immunol. 11,1, 1210. Wybran, J.H. and H.H. Fudenberg, 1971, Trans. Ass. Am. Phys. 84, 239.